PROJECT SUMMARY It is largely unknown how biological complexity of organisms is achieved. Most cellular processes are carried out by proteins through interaction with other proteins. It has been thought that the information that determines protein-protein interactions is encoded within the protein itself. Therefore, it was initially surprising to find that the number of protein-encoding genes and the coding region length have remained fairly constant during evolution from worms to humans. However, the number of genes that produce alternative 3' untranslated regions (3'UTRs) has doubled and 3'UTR length has increased ten-fold during evolution from worms to humans. Furthermore, we recently discovered that 3'UTRs can mediate protein- protein interactions. We found that long 3'UTRs can act as scaffolds that bind RNA-binding proteins, which recruit effector proteins to the site of translation. During translation, the effector protein is transferred from the mRNA to the nascent protein, resulting in the formation of a protein complex that requires the presence of the long 3'UTR. Generalization of this finding suggests that, in the case of alternative 3'UTRs, translation of the short 3'UTR isoform generates the `naked' protein, which finds its protein interaction partners based on random encounters and will bind to the partner with the highest affinity in its surroundings. During translation of the long 3'UTR isoform, however, RNA-binding proteins serve as recruiters for a diverse set of effector proteins, including chaperones, to achieve alternative protein folds, enzymes that add alternative post- translational modifications, or protein binding partners that interact with the nascent protein to form alternative protein complexes. Thus, we propose that 3'UTRs can substantially increase the number of protein-protein interaction partners and may considerably diversify protein functions. With respect to the mechanism of transfer of effector proteins from the RNA to the nascent protein, we hypothesize that mRNAs with long 3'UTRs that are bound by many RNA-binding proteins may nucleate RNA granules whose hydrophobic milieu seems to facilitate electrostatic interactions, thus enabling the transfer of effector proteins. To identify the protein interactors that are recruited by 3'UTRs, we are developing a method called UTR-co-IP. We will transfect cDNA constructs of GFP fusions with the coding region of a candidate gene that either contains no 3'UTR, or its corresponding short or long 3'UTR. GFP-bound proteins will be obtained by co-immunoprecipitation and quantified using mass spectrometry. This will be the first step in investigating if alternative 3'UTRs may contribute to the emergence of biological complexity. Through 3'UTR-mediated RNA granule formation, they enable compartmentalization, through recruitment of binding partners they increase cooperativity, and through the generation of alternative 3'UTRs they facilitate multi-functionality of proteins.